1. Field of the Invention
The present invention relates to a method for producing hair microfollicles by co-culturing the de novo papillae with another cell population of the hair follicle in the ultra-low attachment culture vessels. The invention also relates to a method for producing de novo papillae usable in a method for producing hair microfollicle. Object of the invention are also the hair microfollicles and the de novo papillae produced by the aforementioned methods. The hair microfollicles and/or de novo papillae can be used as implants for treating reduced hair conditions, and for in vitro testing hair-modulating effects or toxic effects of substances.
2. Description of the Related Art
Because of the critical role that hair plays in human non-verbal communication, an affected individual invariably demands help when hair growth diminishes. The ultimate therapy, of course, is to restore or regenerate new, healthy, cycling hair follicles. Until very recently, medicine was unable to offer any valid treatments to these patients. In the late twentieth century, several drugs were marketed that, however modestly and inconsistently, did stimulate hair growth. Examples are minoxidil, finasteride, and latanoprost. The complex timing and myriad gene expression changes required for orchestration of hair follicle development and cycling are likely to preclude a simple pharmaceutical approach to the treatment of advanced alopecia. Consequently, their effects fall short of the ultimate goal to generate new hair follicles in bald scalp. By taking advantage of cell types that know how to form a hair follicle, cell-based therapies will arrive in the clinic sooner than the purely molecular approach.
One approach to hair follicle cell-based therapy would entail removing a small number of hair follicles, isolating competent and/or inductive cells from them, and then expanding those cells ex vivo while maintaining their special ability to generate new hair follicles. Clearly, cell culture conditions that maintain the inductive ability of dermal follicular cells and the competence of hair follicle epithelial cells are necessary before any type of cell-based therapy for alopecia can be developed. As early studies showed that the inductive property of dermal cells wanes with time in vitro, research has focused on maintaining the trichogenic properties of hair follicle cells in culture. Much of the recent progress has resulted from advances in culture methodology for intact hair follicles, and their cellular components. Human hair follicle cells grown in culture include follicular papilla fibroblasts, outer root sheath (ORS) keratinocytes and germinative epidermal cells from the hair matrix (Tobin et al., J. Invest. Dermatology 104(1), 86-88, 1995).
The goal of current bioengineering efforts is to generate or reconstitute fully organized and functional organ systems starting from dissociated cells that have been propagated under defined tissue culture conditions. It has long been recognized that the hair follicle has profound regenerative ability, in that it cycles over the life-time of the individual and reproduces its lower half cycle after cycle. The fibroblasts of the dermal hair papilla and connective tissue sheath are stem cell-like in character and have specific hair growth-inducing properties. The hair follicle reforms itself by means of interactions between competent epithelial stem cells and powerfully inductive dermal cells during its growth cycle. It is possible to reconstitute a complete hair follicle from epithelial and mesenchymal stem cells of hair follicles.
Major challenges that need to be addressed with any type of cell-based treatment for alopecia include the efficiency of hair follicle formation and the choice of cell type, which are summarized by Stenn & Cotsarelis, Curr. Opinion Biotech. 16, 493-497, 2005. For bioengineering the hair follicle, one could start with dermal elements from dissociated follicles with or without competent cells from the follicle or other epithelial sources. The number of dissociated cells would be expanded in culture, and then dermal cells alone, or in combination with competent epithelial cells, re-introduced to the alopecic scalp. Previous studies have shown that starting with correctly placed inducer dermal cells will result in new follicle formation. Moreover, starting with a combination of dissociated, or aggregated, trichogenic epithelial and dermal cells has also proven to be an efficient way of producing new hair follicles.
First attempts at cell-based approaches for treating alopecia are likely to use autologous tissue for bioengineering hair follicles to avoid immune rejection of the donor cells. However, the intriguing possibility that heterologous (allogeneic) hair follicle tissue could be developed for tissue transplantation exists, based on the concept that the hair follicle is an immune-privileged site that does not express MHC (major histocompatibility complex) class I antigens. Nevertheless, the safety testing and regulatory hurdles for this type of approach would require enormous financial resources.
Another possible approach for bioengineering hair follicles involves actually forming hair follicles as mini organs in vitro, and then transplanting the newly generated follicles back to the alopecic scalp. It is known from DE 101 62 814 B4 that a skin and hair equivalent can be produced by providing a pseudodermis or a pseudodermis preparation, as well as pseudopapillae comprising cultivated dermal papilla cells on a suitable carrier or in a suitable matrix, or pseudopapillae precursors comprising cultivated dermal papilla cells on a suitable matrix-forming medium, capable of forming a matrix in situ, and introducing the pseudopapillae or the pseudopapillae precursor into the pseudodermis or pseudodermis preparation. This sort of approach would require a much more complicated cell culture system involving three-dimensional matrices, perhaps embedded with appropriate growth factors, to allow both dermal and epidermal cells to differentiate towards the three-dimensional structure of a normal hair follicle. Particularly, the papilla structure is only obtained by forming cavities, such as by punching or pricking, in said pseudodermis, and placing said pseudopapillae therein, which are so shaped that their dimensions correspond to the cavities formed in the PD. The approach lacks the voluntary arrangement of cells in the approximate physiological papilla structure, as well as direct cell contacts.
It has been recently shown another method for producing a population of multipotent stem cells or progeny thereof, which are originated from a hair follicle or a dermal papilla-containing portion thereof. The method of WO 2005/071063 A1 comprises the culture of said hair follicle or dermal papilla-containing portion in conditions under which multipotent stem cells grow and proliferate non-adherently. Dermal papillae are not obtained by this procedure, but the isolated multipotent stem cells directly used for inducing hair growth or regenerating skin in a mammal. However, it is also recognized in this document that a certain amount of adherent cells is formed as confirmed in WO 2005/113747 A2, which discloses a method for producing multicellular aggregates from at least two multipotent or pluripotent adult stem cell types by cultivation under steric conditions. Presently, the organoid bodies are restricted to the aforementioned stem cells gathered from exocrine gland tissue.